CCR5 is critical for the entry of M-tropic HIV-1 strains in vivo. As CCR5 expression levels in HIV-1 target cells is likely to influence HIV-1 entry, our long-range goal is to define the molecular mechanisms involved in regulating CCR5 gene expression. The rationale is that modulation of CCR5 on cell surfaces is one means of directly affecting CCR5/HIV interactions. Therefore, regulation of CCR5 production, which begins with its constitutive/stimulated transcription, must be understood before it can be determined whether or not these regulatory pathways can be targeted for therapeutic purposes. The first hypothesis to be tested is that the constitutive production of CCR5 is mediated by the interactions of trans-acting proteins and/or protein complexes with the cis-elements located in the two functional promoters that we have recently characterized. In preliminary studies we also show that these two promoters initiate novel, multiple, alternatively spliced CCR5 transcripts. In specific aim 1 we will identify the constellation of DNA response elements and protein/DNA interactions that regulate CCR5 expression. In addition to surrogate cellular milieus, i.e., cell lines, we will study CCR5 transcription in novel physiologically relevant ex-vivo cellular models that we have developed, i.e., human CD34plus progenitor cell-derived monocytes/dendritic cells. The second hypothesis is to be tested is that the magnitude of CCR5 gene transcription and protein synthesis are genetically determined and that the polymorphisms/mutations that we have identified in the regulatory regions of CCR5 account for the observed variability in CCR5 surface expression in individuals who display the CCR5 plus/plus and plus/delta32 genotype. The rationale here is that individuals with different genetic variants of the CCR5 regulatory regions will display different CCR5 surface expression levels. Variable CCR5 levels may directly influence efficiency of infectability with HIV-1, and hence may have important consequences for HIV-1 pathogenesis. To test this hypothesis, in specific aim 2, we will determine the effect of the naturally occurring mutations in the regulatory regions of CCR5 in modulating its transcription, cell surface expression, and HIV-1 coreceptor activity. The proposed experiments offer the potential for identifying novel regulatory mechanisms that control CCR5 expression which could be targeted for developing anti-HIV-1 therapies.